The present invention relates to a novel composition and novel method for detecting an infection by hepatitis B virus (HBV).
Although many viruses can infect human liver, the major human viral hepatitides are caused primarily by infection with hepatitis viruses (types A, B, non-A, and non-B). These viruses cause acute and chronic liver disease which results in significant morbidity and mortality. Hepatitis B, which has a worldwide distribution is the most prevalent form in adults. In addition to acute and chronic liver disease, chronic hepatitis B virus infection can lead to hepatocellular carcinoma, in areas of the world where infection is endemic.
Since hepatitis B virus is medically important, significant effort has been made to understand the biology of the virus and develop diagnostic tests and prophylactic measures. Hepatitis B virus is a DNA virus. Infectious sera or plasma from individuals with hepatitis contain 22 nm spherical and filament particles that do not contain DNA and represent free virus envelopes. Infectious hepatitis B virus is the 42 nm "Dane" particle, which consists of an envelope and a 27 nm nucleocapsid containing DNA. Free nucleocapsids can be observed in the nucleus of infected hepatocytes but are not found in plasma.
Hepatitis B virus possesses several different major antigens which elicit an immune (antibody) response to the virus and form the basis for long term protective immunity. These include hepatitis B surface antigen (HBsAg}, the major envelope antigen, which is found on both the 22 nm and Dane particles. Another surface antigen, termed preS is also found on intact virions and envelopes in serum. Although present in very low abundance, this antigen appears to have significant biologic importance. Recent studies have suggested that hepatitis B vaccines would be more effective if the vaccine formulation also included preS. Since antibodies against HBsAg are protective against hepatitis B infection, this antigen has been intensely studied. Current prophylactic measures are directed toward developing vaccines based on HBsAg.
The genome of hepatitis B virus is a small circular partly double-stranded DNA molecule with a single stranded region of variable length. The long (L) strand is linear and has a fixed length of approximately 3200 nucleotides. The short strand is variable in length from 50 to 75% of L strand. Four open reading frames, termed regions S, P, C, and X, have been identified on the L strand.
The S region which encodes the surface proteins of the virus encompasses nucleotide 2848 to nucleotide 833 in the hepatitis B virus genome. The entire region has the potential for directing the synthesis of a protein 389-400 amino acids in length. HBsAg, the predominant envelope component in both infectious virus and 22 nm particles is a 226 amino acid protein encoded by gene S which begins with the methionine codon at nucleotide 155 and ends at nucleotide 833. Two other methionine codons precede the one at nucleotide 155 in the S region. These methionine codons define the limits of preS2 and preS1 genetic regions that code for preS polypeptides which contain 55 (preS2) and 108-119 (preS1) amino acids. preS1 is located in the hepatitis B virus genome to the 5' side of preS2. Initiation of transcription at any of these methionine codons within the S region can potentially yield three distinct proteins, all having the same 226 carboxyl terminal amino acids but different amino terminal sequences. The amino acid sequence of the preS1 polypeptide is located to the amino terminal side of the preS2 amino acid sequence.
PreS sequences are present on the surface of hepatitis B virus particles in very low amounts (at most they may constitute up to 10% of the surface antigens on the virion or 22 nm particle). Studies have indicated that preS polypeptides are biologically significant. For example, preS2 polypeptide binds aggregated human albumin and appears to be involved in the binding of hepatitis B virus to hepatocytes. Antibodies to preS polypeptides can prevent hepatitis B virus attachment to hepatocytes.
The amino terminal 26 amino acid portion of preS2, termed preS (120-145), has been synthesized. Antibodies to hepatitis B virus bind to either free preS (120-145) or preS (120-145) chemically attached to .beta.-galactosidase, indicating that the amino terminal portion of preS2 is immunodominant.
Because preS determinants elicit the formation of protective antibodies, it would be highly desirable to produce preS polypeptides in sufficient quantities for use as vaccines and in diagnostic tests for hepatitis B infection. However, production of large quantities of the preS polypeptides by previously disclosed techniques, such as purification from hepatitis B virus envelopes or in vitro peptide synthesis is unfeasible. Although recombinant DNA techniques are being used to produce large amounts of a variety of medically useful proteins, including HBsAg, there are no accounts of such techniques being used to produce preS polypeptides.